Two operators are known to bind Escherichia coli galactose repressor with roughly equal affinity. A study of the control these two operators exert on the two overlapping gal promoters is reported. The experiments rest on a set of mutations specifically constructed to inactivate individual control units of the gal operon and on quantitation of gal promoter activities. Messenger RNAs initiated at one or other of the promoters in a cell-free transcription-translation system were determined by a primer extension assay with synthetic deoxyoligonucleotide primers. The main conclusions are: (i) the classical galactose operator O1, located upstream with respect to the two overlapping promoters is sufficient for negative control of the cAMP activated promoter P1; (ii) complete repression of the second promoter P2, on the other hand, needs the presence of both intact operators O1 and O2. Thus, the two overlapping gal promoters (with only 5 bp separating their respective transcriptional start sites) are both subject to negative control by the galactose repressor. This regulation, however, is exerted by two different mechanisms.